Bottom Line:
Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations.Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases.Surprisingly, these mutations were often subclonal and were frequently lost at relapse.

ABSTRACTInfant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10(-5)) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL.

Figure 2: Recurrently mutated genes detected in 47 cases of infant MLL-R ALL. WGS was performed on 22 leukemic samples (discovery cohort) and targeted capture sequencing of the coding exons of 232 genes on an additional 25 infant MLL-R ALL samples (validation cohort). (a) Recurrent mutations were identified in 21 genes/loci in the combined infant MLL-R ALL cohorts. The * indicates that more than two genes were targeted in this locus (see Supplementary Tables 7, 8, 14, 19 and 24). (b) Distribution of mutated genes across the 47 infant MLL-R cases. For mutations in kinases or in genes in the PI3K/RAS pathway, only those known to confer activation of the pathway are shown. * indicates that more than two genes were targeted within the locus in one or more of the cases with only the first gene in the locus listed. † indicates that the sample lacked a matched non-leukemic sample. ∧ indicates that this sample is an identical twin to 060; the matched non-leukemic sample from 060 was used. # indicates that this sample has a novel MLL-USP2 fusion identified through RNA sequencing. A SNV marked with an open circle designates that the mutant allele is expressed as determined by RNA sequencing and a dash indicate that it is not expressed at the level of detection for our analysis.

Mentions:
Despite the paucity of somatic mutations in the discovery cohort, activating mutations in tyrosine kinase/PI3K/RAS pathways were observed, with recurrent mutations in KRAS (n=4), NRAS (n=2), and non-recurrent mutations in FLT3, NF1, PTPN11, and PIK3R1 (Supplementary Tables 6 and 8). In contrast to the non-silent SNVs where only 48% of the mutant alleles were expressed, 100% of the activating kinase/PI3K/RAS pathway mutant alleles were expressed (Supplementary Table 11 and Supplementary Fig. 5). To extend these results, we sequenced the exons of 232 genes that included all mutated genes identified in the discovery cohort, as well as other genes in the kinase/PI3K/RAS signaling pathways, in a validation cohort (for a list of sequenced genes see Supplementary Notes) consisting of an additional 43 infant ALL cases, of which 25 harbored an MLL-R. Each sample was also analyzed for CNAs by SNP arrays (Online Methods and Supplementary Tables 18–23). Recurrent mutations were identified in 21 genes/gene loci across the combined infant MLL-R ALL cohorts (Fig. 2a and Supplementary Table 24). Importantly, activating mutations were identified in tyrosine kinase/PI3K/RAS pathways in 22/47 (47%) of the infant MLL-R cases (Fig. 2a,b, Supplementary Table 25 and Supplementary Figs. 18 and 19). The tyrosine kinase/PI3K/RAS mutations were observed in association with each of the different types of MLL rearrangements identified in infant ALL (Fig. 2b). In every case analyzed by RNAseq, the activating mutant alleles were expressed irrespective of their MAF (Fig. 2b, Supplementary Table 11). Furthermore, gene set enrichment analysis within the MLL-AFF1 cohort revealed the presence of expression signatures consistent with RAS pathway activation (Supplementary Table 26 and Supplementary Figs. 20–23).

Figure 2: Recurrently mutated genes detected in 47 cases of infant MLL-R ALL. WGS was performed on 22 leukemic samples (discovery cohort) and targeted capture sequencing of the coding exons of 232 genes on an additional 25 infant MLL-R ALL samples (validation cohort). (a) Recurrent mutations were identified in 21 genes/loci in the combined infant MLL-R ALL cohorts. The * indicates that more than two genes were targeted in this locus (see Supplementary Tables 7, 8, 14, 19 and 24). (b) Distribution of mutated genes across the 47 infant MLL-R cases. For mutations in kinases or in genes in the PI3K/RAS pathway, only those known to confer activation of the pathway are shown. * indicates that more than two genes were targeted within the locus in one or more of the cases with only the first gene in the locus listed. † indicates that the sample lacked a matched non-leukemic sample. ∧ indicates that this sample is an identical twin to 060; the matched non-leukemic sample from 060 was used. # indicates that this sample has a novel MLL-USP2 fusion identified through RNA sequencing. A SNV marked with an open circle designates that the mutant allele is expressed as determined by RNA sequencing and a dash indicate that it is not expressed at the level of detection for our analysis.

Mentions:
Despite the paucity of somatic mutations in the discovery cohort, activating mutations in tyrosine kinase/PI3K/RAS pathways were observed, with recurrent mutations in KRAS (n=4), NRAS (n=2), and non-recurrent mutations in FLT3, NF1, PTPN11, and PIK3R1 (Supplementary Tables 6 and 8). In contrast to the non-silent SNVs where only 48% of the mutant alleles were expressed, 100% of the activating kinase/PI3K/RAS pathway mutant alleles were expressed (Supplementary Table 11 and Supplementary Fig. 5). To extend these results, we sequenced the exons of 232 genes that included all mutated genes identified in the discovery cohort, as well as other genes in the kinase/PI3K/RAS signaling pathways, in a validation cohort (for a list of sequenced genes see Supplementary Notes) consisting of an additional 43 infant ALL cases, of which 25 harbored an MLL-R. Each sample was also analyzed for CNAs by SNP arrays (Online Methods and Supplementary Tables 18–23). Recurrent mutations were identified in 21 genes/gene loci across the combined infant MLL-R ALL cohorts (Fig. 2a and Supplementary Table 24). Importantly, activating mutations were identified in tyrosine kinase/PI3K/RAS pathways in 22/47 (47%) of the infant MLL-R cases (Fig. 2a,b, Supplementary Table 25 and Supplementary Figs. 18 and 19). The tyrosine kinase/PI3K/RAS mutations were observed in association with each of the different types of MLL rearrangements identified in infant ALL (Fig. 2b). In every case analyzed by RNAseq, the activating mutant alleles were expressed irrespective of their MAF (Fig. 2b, Supplementary Table 11). Furthermore, gene set enrichment analysis within the MLL-AFF1 cohort revealed the presence of expression signatures consistent with RAS pathway activation (Supplementary Table 26 and Supplementary Figs. 20–23).

Bottom Line:
Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations.Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases.Surprisingly, these mutations were often subclonal and were frequently lost at relapse.

ABSTRACTInfant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10(-5)) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL.